Few data on the influence of intratumoral lymphatic microvessel density LMVD on survival in endometrial cancer are available.. Our aim was to assess the intratumoral LMVD of endometrial
Trang 1R E S E A R C H Open Access
Association between intratumoral lymphatic
microvessel density (LMVD) and clinicopathologic features in endometrial cancer: a retrospective cohort study
Lecy Kawamura1, Filomena M Carvalho2*, Bernardo GL Alves2, Carlos E Bacchi3, Joao Carlos Sampaio Goes4, Marcelo Alvarenga Calil4, Edmund C Baracat1, Jesus P Carvalho1
Abstract
Background: Lymph node metastasis in endometrial cancer significantly decreases survival rate Few data on the influence of intratumoral lymphatic microvessel density (LMVD) on survival in endometrial cancer are available Our aim was to assess the intratumoral LMVD of endometrial carcinomas and to investigate its association with classical pathological factors, lymph node metastasis and survival
Methods: Fifty-seven patients with endometrial carcinoma diagnosed between 2000 and 2008 underwent
complete surgical staging and evaluation of intratumoral LMVD and other histologic variables Lymphatic
microvessels were identified by immunohistochemical staining using monoclonal antibody against human
podoplanin (clone D2-40) and evaluated by counting the number of immunostained lymphatic vessels in 10 hot spot areas at 400× magnification The LMVD was expressed by the mean number of vessels in these 10 hot spot microscopic fields We next investigated the association of LMVD with the clinicopathologic findings and prognosis Results: The mean number of lymphatic vessels counted in all cases ranged between 0 and 4.7 The median value
of mean LMVD was 0.5, and defined the cut-off for low and high LMVD We identified low intratumoral LMVD in
27 (47.4%) patients and high LMVD in 30 (52.6%) patients High intratumoral LMVD was associated with lesser miometrial and adnaexal infiltration, lesser cervical and peritoneal involvement, and fewer fatal cases Although there was lower lymph node involvement among cases with high LMVD, the difference did not reach significance
No association was seen between LMVD and FIGO staging, histological type, or vascular invasion On the other hand, low intratumoral LMVD was associated with poor outcome Seventy-five percent of deaths occurred in patients with low intratumoral LMVD
Conclusion: Our results show association of high intratumoral LMVD with features related to more localized
disease and better outcome We discuss the role of lymphangiogenesis as an early event in the endometrial
carcinogenesis
Background
Endometrial cancer is the most frequent gynecologic
malignancy in the western world The death rate has
increased during the most recent decades, probably due to
an increase in life span and coexisting medical
comorbidities [1] The most important prognostic factors
in endometrial cancer are histological type, grade, lymph node status, deep myometrial invasion, and stage [2,3] Approximately 72% of endometrial cancers are stage I, 12% stage II, 13% stage III, and 3% stage IV(1) Five year survival is about 90% in disease confined to the uterus, but drops to 60% when lymph nodes are positive [4] Lymph node metastasis is a complex, multi-step biological process initialized by tumor cells, involving stroma invasion, new
* Correspondence: filomena@usp.br
2
Department of Pathology of Faculdade de Medicina da Universidade de
São Paulo, Sao Paulo (SP), Brazil
Full list of author information is available at the end of the article
© 2010 Kawamura et al; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
Trang 2lymphatic vessel formation, and spread through lymphatic
vessels to lymph node Understanding how cancer cells
gain access to lymphatic channels and develop metastases
is of great interest for development of new therapeutic
strategies targeted against members of the signaling
path-ways involved in the lymphangiogenesis process
The growth of lymphatic vessels largely depends on
many growth factors, such as vascular endothelial
growth factor-C and -D (VEGF-C and VEGF-D), platelet
derived growth factor-BB (PDGF-BB) and hepatocyte
growth factor[5] However, in cancer, these known
lym-phangiogenic factors lead to simultaneous stimulation of
angiogenesis and lymphangiogenesis [6] Lymphatic
microvessel density (LMVD) is one of the ways to
evalu-ate lymphangiogenesis It was poor studied due to the
difficulties associated with detecting and characterizing
lymphatic markers Currently, several molecules
specifi-cally expressed in lymphatic endothelial cells, such as
podoplanin, LYVE1, and the homeobox gene prox-1,
have enabled a more precise study of the lymphatic
vas-culature and the molecular mechanisms involved in
lymphangiogenesis [7] LMVD has been investigated in
many tumors, particularly those characterized by
lym-phatic dissemination, such as cervical carcinomas [8-10],
but there are few studies that have evaluated LMVD in
endometrial carcinomas, and results have been
conflict-ing [11,12]
Therefore, the aim of this retrospective study was to
evaluate the relationship between intratumoral LMVD,
as well as other histologic variables, and the incidence
of lymph node metastasis and overall survival of patients
with endometrial cancer following hysterectomy and
lymph node dissection
Methods
Patients
This study retrospectively analyzed patients with
endo-metrial cancer treated at the Instituto Brasileiro de
Con-trole do Cancer (IBCC), a reference Cancer Center in
São Paulo, Brazil, between 2000 and 2008 This cohort
of 57 patients included 28 (49.1%) with stage I disease, 4
(7.0%) with stage II disease, 22 (38.5%) with stage III
disease, and 3 (5.2%) with stage IV disease, classified
according to the 2009 revised International Federation
of Gynecology and Obstetrics (FIGO) system [13] Age
of patients ranged from 46 to 89 years (67.4 ± 10.27 y)
All patients had undergone surgical staging
hysterect-omy with a sample of pelvic lymph nodes The number
of lymph nodes excised ranged from 3 to 49 (21.5 ±
12.44), and they were positive in 22 (38.6%) and negative
in 35 (61.4%) cases Paraortic lymph nodes were
assessed in 27 cases and nine of them were involved by
the neoplasia The follow-up ranged from 6 to 144
months (mean 39.54 ± 29.98 months) Twelve patients
died of their disease between 6 and 108 months after the diagnosis (median 21.5 months) Data about myo-metrial, cervical, adnexal and peritoneal involvement, were retrieved from the medical records This study was approved by the Department of Obstetrics and Gynecol-ogy Scientific Committee of the Faculdade de Medicina
da Universidade de São Paulo, by the Ethical Committee for Research Projects of the Hospital das Clinical da Faculdade de Medicina da Universidade de São Paulo (Comissão de Ética para Análise de Pesquisa - CAPPesq) (process number 0728/08), and by the Ethical Commit-tee for Research of the Instituto Brasileiro de Controle
do Cancer
Tumor Specimens Tumor slides were reviewed independently by two pathologists and were classified according to the criteria
of the World Health Organization (WHO) Classification
of Tumors [14] The Vancouver system was applied to classify the tumors in low and high grades A tumor was considered high grade if it showed at least two of these criteria: 1) predominantly papillary or solid growth pat-tern, 2) 6 or more mitotic figures/10 high power fields,
or 3) severe nuclear atipia [15] Peritumoral lymphovas-cular space invasion (LVSI) was also evaluated for all cases in the whole histological sections A representative peripheral area of the tumor was selected for the con-struction of tissue microarray blocks and immunohisto-chemical study
Construction of tissue microarray (TMA) blocks The tumor areas selected in the slides were marked in the corresponding paraffin donor blocks and one cylin-der of material (2.0 mm in diameter) was punched from each case and mounted into recipient paraffin blocks at
1 mm intervals using a precision microarray instrument (Beecher Instruments, Silver Spring, MD) A grid system was established such that each core had an x and y coordinate reference for sample identification Blocks were sealed at 60°C for 10 m Three μm sections from each TMA block were then prepared using standard techniques and mounted on Starfrost® slides The first histological sections were stained by hematoxilin-eosin and examined to be sure the correct areas were included
Immunohistochemistry analysis Histological sections (3 μm) from TMA blocks were quenched with 3% hydrogen peroxide solution in phos-phate-buffered saline (PBS; Sigma, St Louis, MO) for 20
m to block endogenous peroxidase activity After several washes in PBS, sections were heated in a microwave (Electrolux, 900 W) for 15 m in 0.01 M citrate buffer,
pH 6.0 for antigen retrieval, then cooled at RT for 20 m
Trang 3Sections were then incubated overnight with a 1:400
dilution of monoclonal antibody against human
podo-planin (clone D2-40, Dako, Carpinteria, CA) Podopodo-planin
is a glycoprotein specifically expressed by lymphatic
endothelial cells In those tumor sections with negative
podoplanin staining, adjacent normal-appearing
lympha-tic endothelial cells served as positive internal controls
Quantification of lymphatic microvessel density
Stained TMA histologic sections were analyzed using
standard light microscopy (Nikon, Eclipse 200) Under
low magnification, the most vascularized intratumoral
areas were identified We counted the number of
immu-nostained lymphatic vessels found in 10 hot spot areas
at 400× magnification Only vessels exhibiting typical
morphology (lumen) were considered lymphatic
micro-vessels (Figure 1) The LMVD for each case was
expressed by the mean value (total number of vessels in
10 hot spot microscopic fields/10) The median value of
all the mean LMVD was the cutoff to divide tumors in
high or low LMVD, as suggested by Hall et al.[16]
Statistical analyses
The association between categorical variables and lymph
node status and survival were evaluated using Pearson’s
Chi-square test Kaplan & Meier method was used to
produce time life tables and survival curves
A p-value less than 0.05 was considered significant
The software used was Epi Infotm, Version 3.5.1 [17]
Results
The tumors were classified as endometrioid (44 cases,
77.2%) and non-endometrioid classified as serous (6
cases, 10.5%), clear cell (4 cases, 7%), undifferentiated (2 cases, 3.5%), and mixed (1 case, 1.7%) The association between all the variables with lymph node status and patient death are summarized in Table 1 The mean number of lymphatic vessels counted in each patient’s tumor sample ranged from 0 to 4.7 The median value was 0.5 vessels and defined the value above and below which was considered high and low LMVD, respectively Low LMVD was found in 27 (47.4%) patients, and high LMVD was found in 30 (52.6%) patients We observed that low intratumoral LMVD was associated with poor outcome Seventy-five percent of deaths occurred in patients with low intratumoral LMVD (p = 0.031) The mean survival time observed in the group with low LMDV was 79 months (confidence interval [62 - 96 months]) and the mean survival time for high LMDV group was 129 months (confidence interval [113 - 145 months]) The difference was significant at 5% level The survival curves for both LMDV groups are presented at Figure 2
Low LMVD was also associated with miometrial and adnaexal infiltration, cervical and peritoneal involve-ment Patients below 70 years presented higher LMVD (21/33, 63.3%) than older patients (9/24, 37.5%) although the difference did not reach statistical signifi-cance No association was seen between LMVD and FIGO staging, histological type, vascular invasion, or lymph node involvement (Table 2)
Positive lymph nodes were significantly associated with histological grade (p = 0.001), vascular invasion (p = 0.017), FIGO stage (p < 0.001), myometrial infiltration (p < 0,001), cervical infiltration (p = 0,023), adnaexal involvement (p = 0,023) and peritoneal involvement (p = 0,048) Besides LMVD, death was associated with histolo-gical type (p = 0.010), vascular invasion (p = 0.040), and lymph node involvement (p = 0.025) Mean survival time was 110 months (range of 84 - 135 months) for patients with negative lymph node involvement, and 69 months (range of 48 - 89 months) for patients with positive lymph nodes
Discussion
Lymphatic metastasis is one of the most important prognostic factors for survival of patients with endome-trial cancer as well as for other epithelial malignancies Many clinical and experimental data suggest that migra-tion of tumor cells into the lymph nodes is facilitated by lymphangiogenesis[18,19] Studies evaluating lymphatic neoformation and its role in tumoral migration are facilitated by the recent identification of lymphangio-genic factors and their receptors, and by lymphatic vas-cular markers [5]
The most commonly used markers for immunohisto-chemistry identification of lymphatic endothelial tissue
Figure 1 Immunohistochemistry staining of a histological
section of endometrioid carcinoma using D2-40 antibody
against podoplanin showing six lymphatic vessels After
counting 10 high power fields, the mean was calculated for each
case (Original magnification X400).
Trang 4include the vascular endothelial growth factor receptor 3
(VEGFR-3), PROX-1, LYVE-1, and podoplanin [5]
Among these, podoplanin, a 38 kDa mucin-type
trans-membrane glycoprotein which is recognized by the
monoclonal antibody D2-40, is considered the most
reli-able marker with a high specificity and sensitivity [5] In
this study, we chose to evaluate podoplanin because it
was found to be expressed in lymphatic, but not blood
vascular endothelium [20], and is expressed in smaller
lymphatic vessels rather than larger ones [20], allowing
a more sensitive identification of lymphangiogenesis
We then considered which compartment (intra or
peritumoral) to evaluate for lymphangiogenesis in this
study, as this is a source of great controversy in the
lit-erature Since our knowledge of lymphangiogenesis in
different types of cancer is still limited, we decided that
it would be worthwhile to begin accumulating data We
believe that a better understanding of the role of
lym-phangiogenesis in tumoral dissemination will only be
achieved after contributions from different researchers groups
In this study, we evaluated LMVD in the intratumoral compartment using podoplanin as the lymphatic marker Vascular density was determined by direct counting of vessels in 10 high power microscope fields Although our study is limited by the fact that LMVD was assessed only in TMA sections, the area of the tumor that was investigated was rigorously chosen considering fixation conditions of the specimen, morphology of the tumor, and the peripheral localization of the sample Besides, our cores had 2.0 mm in diameter, bigger than the con-ventional cores
LMVD has been studied in several cancers by various methods [21] There are few studies about LMVD in gynecological cancer Gombos et al [9] studied intra-and peritumoral LMVD using D2-40 immunohisto-chemistry in 111 cervical squamous cell carcinomas and correlated them with vascular endothelial growth factor
Table 1 Clinicopathological variables and association with lymph node status and outcome
negative n
positive n
alive n
dead n
1
p value (Chi-square of Pearson); 2
Intratumoral lymphatic microvessel density.
Trang 5(VEGF)-C expression, clinicopathologic tumor features,
and outcome They found only high peritumoral LMVD
to be associated with poor overall survival Zhang et al.,
using LYVE-1 (lymphatic vessel endothelial hyaluronan
receptor-1) as the lymphatic marker, studied intra- and
peritumoral lymphatic vascular density in early-stage
invasive cervical carcinomas Both intravascular and
peritumoral high LMVD were associated with lymph
node metastasis [8] In a conflicting study, Birner et al
studied 95 cases of stage pTIb cervical carcinomas and
observed a more favorable prognosis among tumors
with high LMVD [10], a result that is similar to ours
LMVD studies in endometrial cancer are extremely
limited To investigate whether increased peritumoral
and intratumoral LMVD was a good prognostic factor
for nodal metastasis, Gao et al studied 102 patients
with endometrial carcinoma using LYVE-1 as the
lym-phatic marker [22] They found peritumoral but not
intratumoral high LMVD to be associated with lymph
node metastases Vandenput et al studied 62 patients
with endometrial carcinoma and found that neither
peri-tumoral nor intraperi-tumoral lymphangiogenesis determined
by podoplanin expression were related to prognosis [11]
Donughue et al evaluated lymphatic vessel density
(LVD) in 17 cases of endometrial carcinomas by
count-ing vessels expresscount-ing D2-40 Intratumoral LVD was
sig-nificantly increased when compared with endometrial
functionalis LVD, but not when compared with
endo-metrial basalis LVD They also observed a tendency
towards increased intratumoral LVD in grade 3 tumors
compared with grade 1 tumours [23] Other studies have documented lymphatic metastases independent of intratumoral lymphatic vessels suggesting that the intra-tumoral lymphatic vasculature is usually not functioning and only peritumoral lymphatic vessels conduct fluid and cells [24]
In our study, low LMVD was associated with poor outcome Seventy-five percent of deaths occurred in patients with low intratumoral LMVD compared with only 25% of deaths in patients with high LMVD (p = 0.031) In contrast, high LMVD was associated with favorable prognostic factors High LMVD tumors were associated with less myometrial infiltration > 50% (31%
vs 63%, p = 0.034), less cervical involvement (24.1% vs 53.8%, p = 0.023), and less peritoneal involvement (4%
vs 29.4%, p = 0.055) The explanation for this finding is not clear, but we can suppose that lymphangiogenesis is
an early event in tumor progression, when lymphatic vessels are still not functional Considering that higher LMVD was present in early anatomic tumors with no association with intrinsic tumors biological characteris-tics, as histological type or grade, we can suppose that this is an event related to the first steps of local interac-tion between tumor and microenvironment Besides, high LMVD was associated with favorable outcome, indicating that it might be clinically relevant However,
it must be pointed that the vascular density results from
a more complex biologic process that involves a cross-talk between tumor, vessels and host stroma Our results showed higher LMVD among tumor in the initial
Figure 2 Overall survival curve stratified for high and low intratumoral lymphatic microvessel density (LMVD).
Trang 6steps of invasion (no extrauterine extension) and better
prognosis It is well known the role of normal lymphatic
vasculature in maintaining tissue homeostasis [25] The
lymphatic network must be investigated not only by its
anatomic density, as we did, but also functionally We
need intensive further investigation with more early
cases and adequate follow up, to explore the role of
vas-cular density in the tumoral progression These future
results may help elucidate pathways of
lymphangiogen-esis leading to identification of potential drug targets for
endometrial carcinomas Considering our preliminary
results, it is possible that the target would not be the lymphatic vessel itself, but some other molecules involved with its function
Conclusions
Our results show that high intratumoral LMVD is a characteristic of endometrial carcinomas with lesser extrauterine extension and is associated with better out-come However, further studies with larger series are needed to elucidate the biological meaning of these intriguing findings
Acknowledgements
We thank Antonio de Castro Bruni, M.S., for help with statistical analyses, and
Dr Eloa Muniz de Freitas Alves and Prof Joao Guidugli Neto for providing pathological materials This work was supported by grants of the Centro de Estudos do Instituto Brasileiro de Controle do Cancer.
Author details
1 Department of Obstetrics and Gynaecology of Faculdade de Medicina da Universidade de São Paulo, Sao Paulo (SP), Brazil 2 Department of Pathology
of Faculdade de Medicina da Universidade de São Paulo, Sao Paulo (SP), Brazil 3 Consultoria em Patologia, Botucatu (SP), Brazil 4 Instituto Brasileiro de Controle do Cancer, Sao Paulo (SP), Brazil.
Authors ’ contributions
LK collected the data and participated in pathological analyses of the samples and the drafting of the manuscript FMC conceived of the study, participated in its design and review the slides, and helped to the coordination and drafting of the manuscript BGLA reviewed all the histological samples and performed the immunohistochemical analyses CEB carried out the immunohistochemical reactions and helped their
interpretation JCSP, MAC and ECB participated in the design and the coordination, and helped to draft the manuscript JPC conceived of the study and participated in its design, coordination and drafting All authors read and approved the final manuscript.
Competing interests The authors declare that they have no competing interests.
Received: 13 June 2010 Accepted: 14 October 2010 Published: 14 October 2010
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doi:10.1186/1477-7819-8-89
Cite this article as: Kawamura et al.: Association between intratumoral
lymphatic microvessel density (LMVD) and clinicopathologic features in
endometrial cancer: a retrospective cohort study World Journal of
Surgical Oncology 2010 8:89.
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